Orphan Drug Meeting

Target Health will attend & exhibit at CBI’s Orphan Drug Innovation Summit July 17-18, 2013 at The Doubletree by Hilton at Philadelphia Center City. The summit has thought leaders bringing a global perspective on innovative clinical and commercial strategies to help reach and treat patients the world over who are affected by rare diseases. Warren Pearlson will be representing Target Health at our table top exhibit so please stop by and say hello.

Since its founding, Target Health has been very active in the Orphan Drug space. In addition to assisting in an NDA approval last year for a drug to treat of Exocrine Pancreatic Insufficiency (EPI) due to cystic fibrosis (CF) or other conditions, Orphan Drug designations include:

1. Gaucher Disease – NDA Approved
2. Hereditary angioedema – NDA Approved
3. Debridement in hospitalized patients with 3rd degree burns (EMA approved)
4. Burn progression in hospitalized patients
5. Caries prevention, head and neck cancer
6. Cushing’s syndrome secondary to ectopic ACTH secretion
7. Edema-related effects in hospitalized patients with 3rd degree burns
8. Growth Hormone
9. Osteonecrosis of the jaw
10. Systemic Sclerosis (Scleroderma)
11. Alagille Syndrome

For more information about Target Health contact Warren Pearlson (212-681-2100 ext. 104). For additional information about software tools for paperless clinical trials, please also feel free to contact Dr. Jules T. Mitchel or Ms. Joyce Hays. The Target Health software tools are designed to partner with both CROs and Sponsors. Please visit the Target Health Website.

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Gram Staining & Bacteria

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Gram Negative Stained Bacteria
Gram Positive Stained Bacteria

A Gram stain of mixed Staphylococcus aureus (Staphylococcus aureus ATCC 25923, Gram positive cocci, in purple) and Escherichia coli (Escherichia coli ATCC 11775, Gram negative bacilli, in red), the most common Gram stain reference bacteria

 

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Bacteria are 1) ___ by gram staining. This is a process that determines many things resistance to antibiotics, for one thing. It is also one of the first things scientists will do when trying to identify an unknown bacteria. It involves staining a group of bacteria with four different liquids. First, crystal violet is added. Then it is stained with iodine, and finally with safranin. Then it goes through an alcohol wash. The bacteria that retain the purple stain from the crystal violet are gram-positive, and those that take on the pink stain from the safranin are gram-negative.

This has to do with the outer 2) ___ of the cell. The gram-positive bacteria have a thick layer of peptidoglycan, which absorbs the gram stain. Gram-negative bacteria have a thick lipid bilayer on the outside, which is selectively permeable. Not everything can pass through it, and one of those things is the gram stain. The gram-positive bacteria (since things can pass through it easily) are much more susceptible to antibiotics than gram-negative bacteria.

Gram-negative bacteria lose the crystal violet stain (and take the color of the red counterstain) in Gram’s method of staining. This is characteristic of bacteria that have a cell 3) ___ composed of a thin layer of a particular substance (called peptidoglycan). Most bacterial phyla are Gram-negative, including the cyanobacteria, spirochaetes and green sulfur bacteria, and most Proteobacteria.

The Gram-negative bacteria include most of the bacteria normally found in the gastrointestinal 4) ___ that can be responsible for disease as well as gonococci (venereal disease) and meningococci (bacterial meningitis). The organisms responsible for cholera and bubonic plague are Gram-negative.

The Danish bacteriologist J.M.C. Gram (1853-1938) devised this method of staining bacteria using a dye called crystal (gentian) violet. Gram’s method helps distinguish between different types of bacteria. The gram-staining characteristics of bacteria are denoted as positive or 5) ___, depending upon whether the bacteria take up and retain the crystal violet stain or not. For example, Typhus bacillus, is gram-negative, because it doesn’t retain the violet stain.

Gram staining differentiates bacteria by the chemical and physical properties of their 6) ___ walls by detecting peptidoglycan, which is present in a thick layer in Gram positive bacteria. A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color. The Gram stain is almost always the first step in the identification of a bacterial organism. While Gram staining is a valuable diagnostic 7) ___ in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to Gram-variable and Gram-indeterminate groups as well.

Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large 8) ___ (Gram-positive and Gram-negative) based on the physical properties of their cell walls. Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of limited use in environmental microbiology, where some organisms are Gram-variable (that means, they may stain either negative or positive). Some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, Today, much identification is done using genetic 9) ___ and other molecular techniques, which are more specific and informative than differential staining.

Gram stains are performed on body fluid or biopsy when 10) ___ is suspected. Gram stains yield results much more quickly than culture, and is especially important when infection would make an important difference in the patient’s treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.

Gram-positive bacteria have a single, 11) ___ mesh-like cell wall made of peptidoglycan (50-90% of cell envelope), which is stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell envelope), which is stained pink by the counter-stain.

Historically, the Gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

There are four basic steps of the Gram stain:
· Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixing kills some bacteria but is mostly used to affix the bacteria to the slide so that they don’t rinse out during the staining procedure.
· The addition of iodine, which binds to crystal violet and traps it in the cell,
· Rapid decolorization with alcohol or acetone, and
· Counterstaining with safranin. Carbol fuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl-) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I-or I-3) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and, therefore, color the cell. When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell will lose its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV-I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds). After decolorization, the Gram-positive cell remains 12) ___ and the Gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.

Gram-variable
Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative. In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the 13) ___. Normally, if Gram’s stain is done on acid-fast bacteria they will show up as if they are Gram-positive, mostly because of their thick cell wall.

Gram-indeterminate bacteria
Gram-indeterminate 14) ___ do not respond to Gram staining and, therefore, cannot be determined as either Gram-positive or Gram-negative. Examples include Gram-variable and acid fast bacteria.

ANSWERS: 1) classified; 2) layer; 3) wall; 4) tract; 5) negative; 6) cell; 7) tool; 8) groups; 9) sequences; 10) infection; 11) thick; 12) purple; 13) stain; 14) bacteria

 

Hans Christian Joachim Gram (1853-1938)

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Hans Christian Joachim Gram, a Danish bacteriologist, was the son of Frederik Terkel Julius Gram, a professor of jurisprudence, and Louise Christiane Roulund. Gram studied botany at the University of Copenhagen and was an assistant in botany to the zoologist Japetus Steenstrup. His study of plants introduced him to the fundamentals of pharmacology and the use of the microscope. He entered medical school in 1878 and graduated in 1883. His initial work concerned the study of Red blood cells in men. He was among the first to recognize that macrocytes were characteristic of pernicious anemia.

In 1884, while working in Berlin, accidentally stumbled on a method which still forms the basis for the identification of bacteria. While examining lung tissue from patients who had died of pneumonia, he discovered that certain stains were preferentially taken up and retained by bacterial cells. With a few years, Gram developed a staining procedure which divided almost all bacteria into two large groups – the Positive and Negative – Purple and Pink. This staining procedure, devised more than a century ago still serves as one of the most serious procedures for bacterial classification. Today, Microbiologists still feel grateful to the Danish physician Christian Gram, who invented the gram-staining method in 1884.

To do a gramstain, an aviculturist smears a sample of bacteria on a slide, fixes it with heat, and soaks it in a violet dye. Then rinses the slide and soaks it with iodine. The slide is rinsed again with water, then with alcohol or discolorization and counterstained with a pink dye called safranine for a moment. The cell walls of gram-negative bacteria have a very low tolerance for the violet stain, which is decolorized by the alcohol.

Once counterstained with safranine, the gram-negative bacteria appear bright pink to red. Gram-positive cell walls have a high tolerance for the violet stain, and retain it even through the alcohol or decolorizing rinse. When the process is complete, they appear dark purple to brown. The difference between the two cell types appears to be in the amount of peptidoglycan in the cell wall. Gram positive cell walls are about five times as rich in peptidoglycan as gram-negative cell walls. Gram-negative bacteria have a second membrane (chemically different from the plasma membrane) external to the cell wall, and may also have a gelatinous sheath external to the second membrane.

The differences in the cell wall are more than simply a classification/diagnosis tool. Cell wall characteristics are intimately related to the disease-causing potential of the bacterium. In fact, medical researchers have found that an extremely effective way to combat bacterial pathogens is by interfering with cell wall formation. Because the eukaryotic cell has no analog to the prokaryotic cell wall, medicines which target bacterial cell walls have little or no effect on plant or animal cells.

Urine Test Can Diagnose, Predict Kidney Transplant Rejection

 

Following a kidney transplant, patients receive therapy to prevent their immune systems from rejecting the organ. Even with this immunosuppressive therapy, approximately 10-15% of kidney recipients experience rejection within one year after transplantation. Typically, a biopsy is performed only after a transplant recipient shows signs of kidney injury. Although the procedure seldom causes serious complications, it carries some risks, such as bleeding and pain. In addition, biopsy samples sometimes do not give doctors an accurate impression of the overall state of the kidney because the samples are small and may not contain any injured tissue.

According to an article published in the New England Journal of Medicine (2013; 369:20-31) it was reported that an analysis of three biomarkers in the urine of kidney transplant recipients can diagnose-and even predict — transplant rejection. The clinical trial was sponsored by the National Institute of Allergy and Infectious Diseases (NIAID). This test for biomarkers — molecules that indicate the effect or progress of a disease — offers an accurate, noninvasive alternative to the standard kidney biopsy, in which physicians remove a small piece of kidney tissue to look for rejection-associated damage.

According to the NIH, the development of a noninvasive test to monitor kidney transplant rejection status is an important advance that will allow for early intervention to prevent rejection and the kidney injury it causes, which should improve long-term outcomes for transplant recipients. By tracking a transplant recipient’s rejection status over time, it may be possible to modulate doses of immunosuppressive drugs to extend the survival of the transplanted kidney.

In the study, part of the NIH-funded Clinical Trials in Organ Transplantation (CTOT), investigators at five clinical sites collected urine samples from 485 kidney transplant recipients from three days to approximately one year after transplantation. Results of a statistical analysis showed that a group of three urinary biomarkers formed a diagnostic signature that could distinguish kidney recipients with biopsy-confirmed rejection from those whose biopsies did not show signs of rejection or who did not undergo a biopsy. The biomarkers include two messenger RNA molecules that encode immune system proteins implicated in transplant rejection and one noncoding RNA molecule that participates in protein production. The authors used the signature to assign values to each urine sample and identify a threshold value indicative of rejection. With this test, they could detect transplant rejection with a high level of accuracy. The investigators obtained similar results when they tested a set of urine samples collected in a separate CTOT clinical trial, thereby validating the diagnostic signature.

To determine whether the urine test also could predict future rejection, the authors analyzed trends in the diagnostic signature in urine samples taken in the weeks before an episode of rejection. The values for patients who experienced rejection increased slowly but steadily leading up to the event, with a characteristic sharp rise occurring approximately 20 days before biopsy-confirmed rejection had occurred. In contrast, the values for patients who did not show any clinical signs of rejection remained relatively constant and under the threshold for rejection. These findings suggest that it might be possible to treat impending rejection before substantial kidney damage occurs.

Altered Protein Shapes May Explain Differences in Some Brain Diseases

 

Studies have suggested that just one rogue protein (in this case, a protein that is misfolded or shaped the wrong way) can act as a seed, leading to the misfolding of nearby proteins. According to a study published in Cell (3 July 2013), various forms of these seeds — originating from the same protein — may lead to different patterns of misfolding that result in neurological disorders with unique sets of symptoms.

An example of such a protein is alpha-synuclein, which can accumulate in brain cells, causing synucleinopathies, multiple system atrophy, Parkinson’s disease (PD), Parkinson’s disease with dementia (PDD), and dementia with Lewy bodies (DLB). In addition, misfolded proteins other than alpha-synuclein sometimes aggregate, or accumulate, in the same brains. For example, tau protein collects into aggregates called tangles, which are the hallmark of Alzheimer’s disease (AD) and are often found in PDD and DLB brains. Findings from this study raise the possibility that different structural shapes, or strains, of alpha-synuclein may contribute to the co-occurrence of synuclein and tau accumulations in PDD or DLB.

The study was designed to see if different preparations of synthetic alpha-synuclein fibrils would behave differently in neurons that were in a petri dish as well as in mouse brains. Results showed that 2 strains of alpha-synuclein with distinct seeding activity in cultured neurons: while one strain (strain A) resulted in accumulation of alpha-synuclein alone, the other strain (strain B) resulted in accumulations of both alpha-synuclein and tau.

The authors also injected strain A or strain B into the brains of mice engineered to make large amounts of human tau, and then monitored the formation of alpha-synuclein and tau aggregates at various time points. Mice that received injections of synuclein strain B showed more accumulation of tau — earlier and across more brain regions — compared to mice that received strain A.

To evaluate their hypothesis, the authors also examined the brains of 5 patients who had PDD, some of whom also had AD. In this small sample, there was evidence of two different structural forms of alpha-synuclein, one in PDD brains and a distinctly different one in PDD/AD brains, supporting the existence of disease-specific strains of the protein in human diseases.

According to the authors, they are just starting to do work with human tissues and they are planning to look at the brains of patients who had Parkinson’s disease, PDD, or DLB to see if there are differences in the distribution of alpha-synuclein strains.

Although the two strains used in this study were created in test tubes, the authors noted that in human brains, where the environment is much more complicated, the chances of forming additional disease-related alpha-synuclein strains may be greater. These different strains not only can convert normal alpha-synuclein into pathological alpha-synuclein within one cell, they also can morph into new strains as they pass from cell to cell, acquiring the ability to serve as a template to damage both normal alpha-synuclein and other proteins. Therefore, certain strains, but not all strains, can act as templates to influence the development of other pathologies, such as tau tangles.

TARGET HEALTH excels in Regulatory Affairs. Each week we highlight new information in this challenging area

 

FDA to Detain Pomegranate Seeds Offered for Import from Goknur of Turkey – Seeds May Contain Hepatitis A virus

The FDA will detain shipments of pomegranate seeds from Goknur Gida Maddeleri Ithalat Ihracat Tic [Goknur Foodstuffs Import Export Trading] of Turkey when they are offered for import into the United States.

This action results from an investigation by the FDA, the Centers for Disease Control and Prevention, and state and local health authorities into a multi-state outbreak of Hepatitis A illnesses associated with Townsend Farms Organic Antioxidant Blend, a frozen food blend containing pomegranate seed mix. The CDC reported that as of June 27, 2013, 127 people were exposed to Townsend Farms Organic Antioxidant Blend. The illnesses have been reported in Arizona, California, Colorado, Hawaii, Nevada, New Mexico, Utah, and Wisconsin. The people who were reported ill in Wisconsin were exposed to the product in California. The CDC reported that the outbreak strain of Hepatitis A virus (HAV), belonging to genotype 1B, was found in clinical specimens of 56 people in seven states. This strain is rarely seen in the Americas but circulates in North Africa and the Middle East.

By combining information gained from the FDA’s traceback and traceforward investigations and the CDC’s epidemiological investigation, the FDA and CDC have determined that the most likely vehicle for the Hepatitis A virus appears to be a common shipment of pomegranate seeds from Goknur used by Townsend Farms to make the Townsend Farms and Harris Teeter Organic Antioxidant Blends that were recalled in June. These seeds were also used by Scenic Fruit Company to make their recently recalled Woodstock Frozen Organic Pomegranate Kernels.

According to FDA, this outbreak highlights the food safety challenge posed by today’s global food system, and how the presence in a single product of multiple ingredients from multiple countries compounds the difficulty of finding the cause of an illness outbreak.

FDA will be working with the firms that have distributed pomegranate seeds from this shipment from Turkey to help ensure that all recipients of these seeds are notified.

Chicken Salad with Blue Cheese & Apple

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Ingredients

4 cups bite-size cooked chicken
1 fresh celery stalk, chopped
1/2 sweet onion, well chopped
1 garlic clove, juiced
1 green apple, diced (about 1/2 inch dices)
2 teaspoons fresh thyme, chopped well
3/4 cup (or 1 cup) blue cheese, crumbled (Maytag Blue or Danish Blue)
2 Tablespoons sour cream
1 teaspoon turmeric
1 teaspoon prepared mustard
1 Tablespoon green Tabasco sauce (optional)

Directions

Toss together the chicken, celery, onion, apple, turmeric, and thyme in a large bowl.

Dressing:

In a small bowl, gently stir together the blue cheese, sour cream, garlic juice, mustard and Tabasco sauce (optional) in a small bowl. Add the blue cheese dressing to the chicken mixture and gently toss them together. Serve immediately.

This tasty salad is also easy and quick to make. You can use left-over chicken and/or turkey. Or, what I did, was order a barbecued chicken from Dean & Deluca, use all of the white breast meat in the salad, and present legs, & pared down wings to nibble on, with extra blue cheese dressing/dip. This was served with green beans, cauliflower fritters and chilled Chardonnay with green grapes and cheese.

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Enjoy!

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Brave New (Transparent) World

Mark L. Horn, MD, MPH, Chief Medical Officer, Target Health Inc.

The light of full disclosure, it seems, is shining a bit more brightly on the pharmaceutical industry. Much more to follow on this topic.

Our industry, it is becoming clear, is not immune from the transformative changes impacting societal expectations respecting the disclosure of important information. As we witness daily in the political arena, public demands for transparency are increasingly strident and uncompromising. These transcend the established requirements to disclose financial conflicts of interest; the public now wants to know (among other things) how and why key decisions are made, specifics about the processes organizations use, details about ‘perks’ and incentives offered employees, approaches used in training, and the nature and justification of expenses. Virtually nothing is ‘off-limits’. It is indeed a new world.

Under these circumstances, it is unsurprising that the traditional reluctance of the pharmaceutical industry to fully share data with providers and the broader public is being challenged. A recent article in the NYT (“Breaking The Seal On Drug Research”, Katie Thomas, NYT Sunday, June 30, 2013) discussed an investigation by several researchers into the efficacy of Tamiflu®, and the challenges they faced in securing access to the entire dataset of sponsored trials. In a related matter, a duo of studies was recently published in the Annals of Internal Medicine reviewing the efficacy of the biomaterial Infuse® in spinal fusion surgery. The investigators, with the cooperation of the innovator company, were granted access to previously unavailable data which enabled key new insights about safety and efficacy.

Traditionally companies have exercised careful control of their data and had significant leeway in deciding what would be made publicly available. Among other consequences, this behavior has led to the allegation of publication bias, e.g., the predisposition for positive results to selectively make their way into the medical literature. While this may be as much a consequence of editorial preference to publish more interesting, e.g. positive results, as corporate practice, the net result is a risk that the profile of medicines may not be accurately reflected in the literature. This leads to situations like that described in the NYT article; uncertainty among physicians about the efficacy and safety of widely used and much needed medicines.

Traditional arguments favoring limiting access to data, including protecting intellectual property and innovative discovery processes, are proving less compelling to both the public and providers. While threatening to established practice, the demands of increasingly sophisticated and empowered patients and providers make increased data transparency inevitable. The evolving dynamic will likely engage multiple stakeholders: providers, patients and innovators, who will learn about the nuances of new medicines — including how to manage the uncertainty of incomplete information –collaboratively in real time. The benefits are profound, an enhanced ability to pose and answer critically important questions; e.g., is an established chemotherapeutic agent effective against a different cancer type or, how does a new biologic compare with available therapies in treating autoimmune disease? Ultimately all stakeholders, industry included, should benefit from more sophisticated knowledge of the safety and efficacy of the pharmacopoeia. Clinical care should improve, and the societal commitment to ensuring the intellectual property protection required for robust new product development should be enhanced.